Our previous studies indicated that certain pseudo-diploid SS-teratocarcinoma stem cell lines contain two genetically active X-chromosomes as long as they are maintained in the undifferentiated state, and that the cells can undergo X-inactivation when they are allowed to differentiate in vitro. We propose here to further explore the extent to which such cells can provide a valid model system for the study of X-inactivation. To do this, we will isolate new teratocarcinoma stem cell lines by a variety of techniques, including fusion with microcells derived from M. caroli fibroblasts, to obtain cells that are heterozygous for electrophoretic variants of X-linked enzymes. The availability of such cell lines will enable us to confirm by enzyme gel electrophoresis our earlier conclusions. In other experiments we will study the cytogenetic properties of the X-chromosomes in the cells to determine the temporal relationship between the onset of the cytological manifestations of X-inactivation (e.g. late replication of DNA) and of biochemical inactivation. In addition, we will determine whether an inactive X-chromosome can be reactivated by placing it in a teratocarcinoma stem cell.